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Abstract and Introduction

Abstract

The spread of resistance among Gram-positive and Gram-negative bacteria represents a growing challenge for thedevelopment of new antimicrobials. The pace of antibiotic drug development has slowed during the last decade and, especiallyfor Gram-negatives, clinicians are facing a dramatic shortage in the availability of therapeutic options to face the emergency ofthe resistance problem throughout the world. In this alarming scenario, although there is a shortage of compounds reaching themarket in the near future, antibiotic discovery remains one of the keys to successfully stem and maybe overcome the tide ofresistance. Analogs of already known compounds and new agents belonging to completely new classes of antimicrobials are inearly stages of development. Novel and promising anti-Gram-negative antimicrobials belong both to old (cephalosporins,carbapenems, β-lactamase inhibitors, monobactams, aminoglycosides, polymyxin analogues and tetracycline) and completelynew antibacterial classes (boron-containing antibacterial protein synthesis inhibitors, bis-indoles, outer membrane synthesisinhibitors, antibiotics targeting novel sites of the 50S ribosomal subunit and antimicrobial peptides). However, all of thesecompounds are still far from being introduced into clinical practice. Therefore, infection control policies and optimization in theuse of already existing molecules are still the most effective approaches to reduce the spread of resistance and preserve theactivity of antimicrobials.

Introducion

The spread of resistance among Gram-positive and Gram-negative bacteria represents a growing challenge for thedevelopment of new antimicrobials.[1–3] In 2011, multidrug resistance (MDR) and extreme-drug resistance (XDR), defined asnonsusceptibility to one or more antimicrobials in three or more antimicrobial classes and nonsusceptibility to all antimicrobialsrespectively, pose a serious and emerging threat to patients in healthcare settings and have a significant impact on mortality,costs and length of hospital stay.[4–6] Some authors have described this phenomenon with the acronym 'ESCAPE', to explainthe more frequent MDR microorganisms with the first initial of the following bacteria: Enterococcus faecium, Staphylococcusaureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae.[5] In addition,infections due to MDR strains of Escherichia coli, an important pathogen responsible for healthcare-associated and community-acquired infections, are increasingly being reported.[7] The increase in prevalence of resistant strains amongEnterobacteriaceae and nonfermenters in the USA and Europe is shown in Figures 1 & 2. Resistance to currently availabledrugs is a consequence of this phenomenon and, especially for Gram-negatives, clinicians are facing a dramatic shortage in theavailability of therapeutic options. Therefore, although the pace of antibiotic drug development has slowed during the lastdecade, there is a critical need for new antimicrobials (Figure 3). However, until new drugs reach clinical practice, one of theresidual and effective strategies to treat infections caused by MDR Gram-negatives is the optimization in the use of alreadyexisting molecules. For example, the Probability of Target Attainment of Antibacterial Agents Studied for Susceptibility andPharmacodynamic Optimization in Regional Trials (PASSPORT) study demonstrated that, especially for nonfermenting Gram-negative bacilli such as A. baumannii and P. aeruginosa, high-dose prolonged infusions of cefepime, ceftazidime, doripenemand meropenem have higher probabilities of achieving bactericidal exposure compared with standard 30-min infusion regimens,reducing the risk of treatment failure.[8] The progressive erosion of therapeutic options has also forced clinicians to rediscoverold drugs such as polymyxins and fosfomycin. Regarding the development of new antimicrobials, the Infectious DiseasesSociety of America (IDSA) and the US FDA have tried to draw attention to the lack of new antibiotics for MDR Gram-negativebacteria since 2004.[9] Moreover, the IDSA has recently supported the '10 × 20' initiative towards the development of ten newantibacterials by 2020.[10] Although truly new therapeutic options against MDR Gram-negatives are still far from clinical practice,several compounds are now undergoing preclinical or early-phase clinical studies. The issue of resistance to currently used anti-Gram-negative agents and future perspectives about new drugs belonging to both old and new classes of antimicrobials will bediscussed.

Will New Antimicrobials Overcome Resistance AmongGram-negatives?Matteo Bassetti; Francesca Ginocchio; Małgorzata Mikulska; Lucia Taramasso; Daniele RobertoGiacobbePosted: 11/21/2011; Expert Rev Anti Infect Ther. 2011;9(10):909-922. © 2011 Expert Reviews Ltd.

Figure 1. Resistance rates among Gram-negative bacteria in France, Italy, Spain and the UK. Data from [2,3].

Figure 2. Resistance rates among Gram-negative bacteria in the USA. Data from [1].

Figure 3. New antibacterial agents approved in the USA, 1983–2009 (as reported by the Infectious DiseasesSociety of America's Antimicrobial Availability Task Force). Data from [9].

Mechanism of Resistance in MDR Gram-negative Bacteria

Gram-negatives have developed several mechanisms of resistance to currently used antimicrobials, including β-lactamases,efflux pumps, porin mutations, modifying enzymes and binding site mutations. In addition, horizontal transfer of combinedresistance to multiple drugs is partly responsible for the rapid emergence of resistance in human and veterinary medicine.Significantly, the rapid emergence of resistance to various antibiotics is partly due to resistance genes associated in clusters andtransferred together. Horizontal transfer is a successful mechanism for the transmission and dissemination of multiple-drugresistance among bacterial pathogens.[11] The impact of horizontally transmitted resistance mechanisms on clinical practice isremarkable, as indicated by the spread of Klebsiella pneumoniae carbapenemases.[12] The awareness of the most diffuse andrelevant resistance patterns among bacteria is the basis not only for an adequate administration of antimicrobials, but also forthe development of new compounds aiming to overcome specific resistance determinants.

Resistance to β-lactams

Although bacteria may develop several mechanisms of resistance to β-lactams, the most important and efficient method ofresistance to these agents in Gram-negatives is the synthesis of β-lactamases. β-lactamases have presumably evolved to fightnatural β-lactams, produced by bacteria such as Streptomyces or Lysobacter, or filamentous fungi, such as Penicillium orAcremonium.[13] However, the widespread administration of antibiotics has heavily influenced the development of β-lactamase-mediated resistance. Since β-lactam antibiotics came into clinical use, β-lactamases have coevolved with them. One of the firstβ-lactamases able to confer resistance to β-lactams, known as TEM-1 (the enzyme was originally found in E. coli isolated from apatient named Temoniera), developed in the sixties after the widespread use of ampicillin in human medicine.[14] Nowadays,extended-spectrum β-lactamases (ESBLs) and a variety of carbapenemases, which hydrolyze third-generation cephalosporinsand carbapenems respectively, compromise the use of β-lactams in clinical practice.[15] For example, ESBL-producing E. coliand Klebsiella spp. are common in healthcare settings and pandemic clones of ESBL-producers, such as E. coli ST131, alsocause community-acquired infections.[7] Moreover, in a meta-analysis of 16 studies including 1682 infections, bacteremiascaused by ESBL-producing pathogens were significantly associated with delayed initiation of effective therapy and an almosttwofold increase in crude mortality.[16] Some examples of important ESBLs are TEM, SHV and CTX-M β-lactamases.[17] Forexample, class A CTX-M-15 enzyme is frequently harbored by the previously mentioned E.coli ST131.[7] The widely usedAmbler classification of β-lactamases, which divides enzymes into four classes (A, B, C and D) based upon their amino acidsequences, is outlined in Table 1.[17,18] ESBLs are mostly class A enzymes, even though there are ESBLs belonging to otherclasses as well. A resistance phenotype similar to that of ESBLs-producers may be conferred by the overexpression ofconstitutive or inducible AmpC β-lactamases (class C), which are also resistant to inhibition by commercially available β-lactamase inhibitors.[19] This enzyme diversity is a crucial and strategic aspect in the development of new β-lactams and β-

lactamase inhibitors. Carbapenems can be inactivated by β-lactamases belonging to class B (metallo-β-lactamases [MBLs]),class A (K. pneumoniae carbapenemases) and class D (OXA-23, OXA-48). MBL-mediated resistance is common among P.aeruginosa and Acinetobacter spp., nonfermenting pathogens that frequently harbor resistance to multiple antibiotics.[19]

However, resistance to carbapenems often reflects an interplay involving more than one mechanism: carbapenemases or otherβ-lactamases with weak hydrolyzing activity (e.g., AmpC or class D β-lactamases), changes in membrane permeability throughthe loss of specific porins, efflux pumps and structural changes in protein-binding proteins.[20] The emergence of MBL-mediatedresistance among Enterobacteriaceae has also become a serious public health concern. Moreover, the increase in internationaltravel is likely to be a contributory factor for the ascendancy of mobile MBL genes as much as the mobility among individualbacteria. A new plasmidic MBL, the New Delhi MBL called NDM-1, has been recently identified in K. pneumoniae and E. colirecovered from a Swedish patient who was admitted to a hospital in New Delhi, India.[21] Recently, reports from Pakistan, India,the UK and other countries also describe the global spread of NDM-1-producing Enterobacteriaceae, frequently resistant tocurrently used antimicrobials, with the exceptions of colistin and tigecycline.[22,23] A recent US study reported that in 2008 asmuch as 17% of P. aeruginosa and 74% of A. baumannii strains were MDR.[1] Moreover, the incidence of resistance tocarbapenems among Acinetobacter spp. increased from 9% in 1995 to 40% in 2004.[24]

Table 1. Classification schemes for bacterial β-lactamases.

Molecularclass(subclasses)

Bush-Jacobygroup(2009)

Distinctivesubstrate(s) Defining characteristic(s) Representative

enzyme(s)

A 2a Penicillins Greater hydrolysis of benzylpenicillin thancephalosporins PC1

A 2b Penicillins, earlycephalosporins

Similar hydrolysis of benzylpenicillin andcephalosporins

TEM-1, TEM-2,SHV-1

A 2beExtended-spectrumcephalosporins,monobactams

Increased hydrolysis of oxyimino-β-lactams(cefotaxime, ceftazidime, ceftriaxone, cefepime,aztreonam)

TEM-3, SHV-2,CTX-M-15, PER-1,VEB-1

A 2br Penicillins Resistance to clavulanic acid, sulbactam andtazobactam TEM-30, SHV-10

A 2berExtended-spectrumcephalosporins,monobactams

Increased hydrolysis of oxyimino-β-lactamscombined with resistance to clavulanic acid,sulbactam and tazobactam

TEM-50

A 2c Carbenicillin Increased hydrolysis of carbenicillin PSE-1, CARB-3

A 2ce Carbenicillin,cefepime

Increased hydrolysis of carbenicillin, cefepimeand cefpirome RTG-4

A 2e Extended-spectrumcephalosporins

Hydrolyzes cephalosporins. Inhibited byclavulanic acid but not aztreonam CepA

A 2f Carbapenems Increased hydrolysis of carbapenems,oxyimino-β-lactams, cephamycins

KPC-2, IMI-1, SME-1

B (B1) 3a Carbapenems Broad-spectrum hydrolysis includingcarbapenems but not monobactams

IMP-1, VIM-1, CcrA,IND-1

B (B2) 3b Carbapenems Preferential hydrolysis of carbapenems CphA, Sfh-1

B (B3) L1, CAU-1, GOB-1,FEZ-1

C 1 Cephalosporins Greater hydrolysis of cephalosporins thanbenzylpenicillin; hydrolyzes cephamycins

E. coli AmpC, P99,ACT-1, CMY-2,FOX-1, MIR-1

C 1e Cephalosporins Increased hydrolysis of ceftazidime and oftenother oxyimino-β-lactams GC1, CMY-37

D 2d Cloxacillin Increased hydrolysis of cloxacillin or oxacillin OXA-1, OXA-10

D 2de Extended-spectrumcephalosporins

Hydrolyzes cloxacillin or oxacillin and oxyimino-β-lactams OXA-11, OXA-15

D 2df Carbapenems Hydrolyzes cloxacillin or oxacillin and OXA-23, OXA-48

Resistance to Colistin & Tigecycline

Colistin belongs to the antimicrobial class of polymyxins and acts by binding to the lipid A moiety of the bacteriallipopolysaccharide and subsequently disintegrating the bacterial membranes.[23] Resistance to colistin has been associated withlipopolysaccharide modifications leading to a high-level of antibiotic nonsusceptibility.[25] A detailed review of various resistancemechanisms to polymyxins has recently been published.[23]

Tigecycline, the first glycylcycline to be approved by the FDA, may lose its activity against MDR strains, particularly when lowdrug concentrations are attained in the serum during treatment. Resistance develops when the MICs of the targeted pathogenexceed the Cmax of the drug, which is almost the rule for all targeted A. baumannii strains.[26] According to molecular studies,efflux pumps seem to be the most important mechanism of resistance to tigecycline both in Enterobacteriaceae andnonfermenting rods.[27]

Resistance to Fluoroquinolones & Aminoglycosides

Fluoroquinolones such as ciprofloxacin and aminoglycosides such as gentamycin or amikacin are compounds with bactericidalactivity against Gram-negative pathogens. However, resistance to these widely used antimicrobials is increasing. For example,the MDR pandemic clone E. coli ST131 is frequently reported as resistant to fluoroquinolones, as well as to third-generationcephalosporins.[7]

Fluoroquinolones act by binding to the nuclear enzymes DNA gyrase and topoisomerase IV.[28] In spite of their particulartargets, resistance mechanisms, both chromosomal and plasmidic, have been developed. Chromosomal resistancemechanisms include target mutations (GyrA/GyrB for DNA gyrase and ParC/ParE for topoisomerase IV), and augmentedexpression of efflux pumps.[28] Among plasmidic-mediated resistance the most important mechanisms are represented byacetylation, efflux pumps and production of fluoroquinolones resistance proteins (Qnr), which protect the targets frominhibition.[28]

Aminoglycosides inhibit protein synthesis by binding to 16S rRNA and they also disrupt the integrity of the bacterial cellmembrane.[29] There are three different mechanisms responsible for resistance to aminoglycosides: overexpression of effluxpumps, target modification and presence of modifying enzymes.[30]

Resistance to Phosphonic Acid Derivatives

Fosfomycin, a phosphonic acid derivative, inhibits an enzyme involved in the first step of bacterial cell-wall synthesis.Resistance to fosfomycin is rarely reported because of its particular molecular structure and mechanism of action.[31]

Fosfomycin is highly active against ESBL-producing Enterobacteriaceae (among 748 K. pneumoniae and 1657 E. coli isolateswhich produced ESBLs, 81 and 97% were susceptible to fosfomycin, respectively).[32] Therefore, fosfomycin may still representan important alternative in the treatment of urinary tract infections, including those caused by MDR pathogens. However,bacteria can develop resistance to fosfomycin, and the most commonly described resistance mechanism in E. coli is theoverexpression of the plasmidic genes FomA and FomB, leading to phosphorylation of fosfomycin and fosfomycinmonophosphate.[32]

Novel β-lactams & β-lactamase Inhibitors

The research into new drugs able to overcome resistance to β-lactams may adopt two different approaches. The development ofnovel compounds belonging to already know classes of β-lactams such as cephalosporins, carbapenems and monobactamsmay lead to the synthesis of structures with stability to enzymatic hydrolysis, whereas the combination of β-lactams with new β-lactamase inhibitors may protect β-lactams from the action of ESBLs and MBLs. In fact, currently used β-lactamase inhibitorsare highly active only against class A enzymes, while activity against class C and class D is poor and they are not active againstclass B β-lactamases.[33] Reported MICs of different Gram-negative strains to some of the new β-lactamase inhibitors and othernovel compounds belonging to old and new classes of antimicrobials are outlined in Table 2.

carbapenemsData from [17].

Table 2. MIC90 of some new agents and comparators against Gram-negative rods in differentstudies.

Bacteria (number of isolates) MIC90 (range), mg/l Ref.

Novel β-lactam & β-lactamase inhibitors

BLI-489 Piperacillin + tazobactam Piperacillin + BLI-489 [65]

E. coli (52) 2 (0.5–128) 2 (0.25–64)

Novel Cephalosporins

Among fifth-generation cephalosporins, ceftobiprole and ceftaroline demonstrated good activity against methicillin-resistant S.aureus (MRSA), but there is no evidence of an enhanced activity against MDR Gram-negative strains when compared with oldercephalosporins. Following the results of a randomized, double-blind trial published in 2008, ceftobiprole has been approved inSwitzerland and Canada for the treatment of adults with complicated skin and skin-structure infections due to Gram-positive andGram-negative bacteria, but the US and European approval procedures are ongoing.[34] Ceftobiprole has a low potential forinducing chromosomal AmpC β-lactamases but it is hydrolyzed by most ESBLs and MBLs.[35] Ceftaroline was approved by theFDA in 2010 for the treatment of acute bacterial skin and skin-structure infections and community-acquired bacterialpneumonia.[36] It has shown synergistic activity with tazobactam against MDR Gram-negative pathogens such as ESBL-producing E. coli and K. pneumoniae.[37] However, in a Phase III clinical trial, it was less active than currently used antimicrobialagents against Gram-negatives, as a combination of vancomycin plus aztreonam demonstrated higher favorable microbiologicalresponse rates (86.3 vs 93.6%; p = not statistically significant).[38] Moreover, ceftaroline has been reported to have low MICs formany Enterobacteriaceae, similar to those of oxyimino-cephalosporins and, like these comparators, it lost activity against ESBL-producers, with MICs being especially high for isolates encoding CTX-M enzymes.[39] Among 15,011 clinical isolates obtainedfrom patients in Canadian hospitals between 2007 and 2009, ESBL-producing E. coli and K. pneumoniae were not susceptibleto ceftaroline and the in vitro activity of ceftaroline versus P. aeruginosa was similar to that of cefepime (MIC90: 16 µg/ml).[40]

CXA-101, a novel cephalosporin under development as a single agent and in combination with tazobactam (Phase II),represents a compound of particular interest for the treatment of Gram-negative infections. CXA-101 has potent activity againstP. aeruginosa, which is not diminished by AmpC overexpression, porin mutations or efflux pumps.[41,42] Phase I studies showeda favorable safety and predictable pharmacokinetic profile with high target attainment for the urinary tract.[43] CXA-101

E. coli ESBL-A† (31) >128 (1 to >128) 16 (1–32)E. coli AmpC (17) 32 (2–64) 16 (1–16)

E. cloacae (52) >128 (0.5 to >128) 16 (0.5–16)

K. pneumoniae (54) 16 (1 to >128) 8 (1–16)

K. pneumoniae ESBL-A (36) >128 (2 to >128) 2–128 (32)

K. pneumoniae AmpC (30) >128 (8 to >128) >128 (4 to >128)

Acinetobacter spp. (30) 32 (≤0.12 to >128) 16 (0.5–32)

P. aeruginosa (55) >128 (4 to >128) 64 (4 to >128)

ME1071 Meropenem Meropenem + ME1071 [80]

MBL-producing P. aeruginosa (174) >64 (0.5 to >64) >64 (0.25 to >64)

Non MBL-producing P. aeruginosa (16) 64 (0.12–64) 64 (0.5–64)

Tomopenem Meropenem Tomopenem [55]

E. coli (25) ≤0.03 (≤0.03–0.25) ≤0.03 (≤0.03–0.12)

K. pneumoniae (25) ≤0.03 (≤0.03–0.06) 0.06 (≤0.03–0.12)

P. aeruginosa (100) 16 (0.06 to >32) 4 (0.06–32)

Novel polymyxins

CB-182,804 Colistin CB-182,804 [86]

E. coli (80) 0.5 2

K. pneumoniae (81) 2 4

P. aeruginosa (100) 2 2

Acinetobacter spp. (81) 4 4

Protein synthesis inhibitors

AN3365 Imipenem AN3365 [90]

P. aeruginosa (101) >64 (0.25 to >64) 8 (1–16)

Acinetobacter spp. (25) >64 (8 to >64) 16 (4–32)†Class A ESBL.ESBL: Extended-spectrum β-lactamase; MBL: Metallo-β-lactamase.

combined with tazobactam has been evaluated against 169 EBSL-producing strains and showed better in vitro activity thancurrently available β-lactam/β-lactamase inhibitor combinations.[44]

Novel Carbapenems

Carbapenem therapy is often used for serious Gram-negative infections. Compounds belonging to this class of antimicrobialsbind to critical penicillin-binding proteins (PBPs), disrupting the growth and structural integrity of bacterial cell walls. Theyprovide enhanced anaerobic and Gram-negative coverage as compared with other β-lactams and the main advantage ofcarbapenems in the treatment of infections due to MDR Gram-negatives is their stability to hydrolysis by many ESBLs.Nevertheless, as previously reported, several mechanisms of resistance may confer, alone or in synergy, resistance to thesecompounds.[20]

Indeed, the Clinical and Laboratory Standards Institute has recently established revised criteria for the interpretation ofbreakpoints for carbapenems (Table 3).[45] Several novel compounds in clinical development or in preclinical phases arereported in Table 4.

Table 3. New carbapenems' breakpoints for Enterobacteriaceae.

Drug MIC (mg/l)

Susceptible Intermediate Resistant

New carbapenem breakpoints

Doripenem ≤1 2 ≥4

Ertapenem ≤0.25 0.5 ≥1

Imipenem ≤1 2 ≥4

Meropenem ≤1 2 ≥4

Old carbapenem breakpoints

Ertapenem ≤2 4 ≥8

Imipenem ≤4 8 ≥16

Meropenem ≤4 8 ≥16

Data from [45].

Table 4. Status and pharmacokinetic characteristics of new carbapenems.

Drug Status Dose Administration Half-life(h)

Activity against

Enterobacteriaceae Pseudomonasaeruginosa

Acinetobacterspp. MRSA

Doripenem US FDAapproved

500 mgt.i.d. iv. 1 + + + -

Panipenem

Approvedin Japan,ChinaandKorea

0.5/0.5 gb.i.d. iv. 1.1–

0.7 + - ? -

Biapenem Phase II 300 mgb.i.d. iv. 1.03 + + + -

Tomopenem Phase II 700 mg iv. 1.7 + + + +

Razupenem Phase II Not yetestablished iv. ? + + ? +

Tebipenem Phase II 4–6 mg/kgb.i.d. Oral ? + - ? -

+: Active; -: Nonactive; ?: Unknown; b.i.d.: Twice a day; iv.: Intravenous; MRSA: Methicillin-resistant Staphylococcus aureus; t.i.d.: Three times a day.Data from [101].

Doripenem, a parenteral carbapenem with broad-spectrum activity, including methicillin-susceptible S. aureus and coagulase-negative staphylococci has recently been marketed.[46–48] However, its activity against MDR Enterobacteriaceae is somewhatsimilar to that of meropenem.[46–48] Doripenem is stable to hydrolysis by many β-lactamases of classes A, C and D, includingESBLs, with high affinity demonstrated for the AmpC cephalosporinase. However, poor activity against MBLs and serinecarbapenemases of classes A and D has been reported.[49] Doripenem has been compared with imipenem and the drugs seemequivalent, except in patients with P. aeruginosa infections where clinical cure was 80.0% (doripenem) and 42.9% (imipenem; p= not significant).[50]

Biapenem is a novel carbapenem approved in Japan in 2002, which reached a high concentration in respiratory tissue and otherbody fluids. In a multicenter, open-label, randomized controlled clinical study on 216 patients, biapenem was as effective andwell-tolerated as imipenem/cilastatin for the treatment of intermediate and severe bacterial respiratory or urinary tractinfections.[51]

Biapenem has a broad spectrum of activity including Gram-positive and Gram-negative bacteria such as S. pneumoniae,methicillin-susceptible S. aureus, A. baumannii and ESBL-producing Enterobacteriaceae, while a moderate activity (medianMIC: 8 mg/l) was found against P. aeruginosa.[52,53] On the other hand, among three of 19 MBL-producing P. aeruginosastrains, susceptibility to meropenem and biapenem has been reported despite the resistance to imipenem, whereas 16 strainswere resistant to all three antimicrobials.[54]

Tomopenem (formerly CS-023) is a novel carbapenem with broad-spectrum activity against different hospital pathogens,including P. aeruginosa and MRSA. Moreover, it seems to have a very low rate of spontaneous emergence of resistance. Invitro activity against β-lactam-susceptible and -resistant strains of MRSA, ceftazidime-resistant P. aeruginosa and ESBL-producing Enterobacteriaceae has been demonstrated.[55]

Razupenem (previously known as SMP-601) is a novel compound in Phase II of evaluation with in vitro activity against ESBL-producers. However, its activity was significantly reduced by AmpC enzymes and carbapenemases.[56]

Panipenem is a parenteral carbapenem approved in Asia for the treatment of urinary tract infections (UTIs), andobstetrical/gynecological, lower respiratory tract and surgical infections.[57] The combination of panipenem with betamipron, likeimipenem/cilastatin, is necessary. Panipenem/betamipron (the combination is necessary because betamipron inhibits the renaluptake of the carbapenem) demonstrated no inferiority to imipenem/cilastatin in the treatment of adult patients with respiratoryinfections and UTIs in three different large, randomized, Phase III clinical trials.[57] Against 520 aerobic Gram-negativeorganisms, panipenem had comparable activity to imipenem.[58]

ME1036 is a novel parenteral carbapenem, active against Gram-positives and Gram-negatives, including ESBL-producingEnterobacteriaceae. ME1036 has excellent activity against MRSA isolated from blood cultures in patients with bacteremia andcommunity-acquired bacterial pneumonia requiring hospitalization.[59]

Tebipenem pivoxil is a prodrug of an oral carbapenem with no activity against MRSA and MBL-producing pathogens.[46] Itshowed good activity against K. pneumoniae and E. coli isolates.[46] An advantage of tebipenem pivoxil may be the activityagainst Acinetobacter spp., associated with good oral bioavailability.

Of note, P. aeruginosa is not susceptible to three of the carbapenems described (ME1036, panipenem/betamipron andtebipenem).

Novel Monobactams

BAL30072 is a monosulfactam with activity against PBP-producing strains. In vivo and in vitro activity of BAL30072 incombination with meropenem has been tested against five different strains of A. baumannii, three of which were producers ofcarbapenemases. Combining BAL30072 and meropenem (2:1 and 1:1) lowered in vitro meropenem MICs two- to eight-fold and,in vivo, BAL30072 was active against all four strains that were inhibited in vitro by BAL30072.[60]

In another study BAL30072 demonstrated bactericidal activity against both Acinetobacter spp. and P. aeruginosa, even againststrains that produced MBLs.[61]

It was also active against many species of MDR Enterobacteriaceae isolates, including those with class A or Bcarbapenemases.[61]

Novel β-lactamase Inhibitors

There are several potential β-lactamase inhibitors under investigation, in different stages of preclinical and clinical trials.Although the results of Phase I and II studies were mostly not published in peer-reviewed journals, they seem particularlypromising as therapeutic agents. The principal strategy to develop β-lactamase inhibitors able to overcome microbial resistanceto β-lactams and currently used β-lactam inhibitors is to find molecules which inhibit class B, C and D enzymes and restore the

activity of penicillins, cephalosporins or carbapenems against MDR strains. According to their molecular structure, theseinhibitors can be classified as β-lactams and non-β-lactams, whereas the differences between novel compounds active againstESBLs and AmpC β-lactamases and those able to also inhibit MBLs may be of important therapeutic value. The potential activityof these compounds against the different classes of β-lactamases is outlined in Table 5.

Inhibitors Active against ESBL & class C β-lactamases

Extended-spectrum β-lactamases and class C β-lactamases are important vehicles of resistance among Enterobacteriaceaeand nonfermenting Gram negatives.[62] Several novel β-lactamase inhibitors in different phases of clinical trials have shownpromising activity against MDR strains carrying these enzymes.

Novel imidazole-substituted 6-methylidene-penem molecules demonstrated excellent in vitro inhibition of the TEM-1 and AmpCenzymes with significantly higher activity compared with tazobactam, which already has better class C activity than clavulanicacid.[63,64] The compound with the most promising clinical data among these agents is BLI-489. It has shown in vitro activityagainst class A, C and D enzymes and the activity of piperacillin against some ESBL-producing strains nonsusceptible topiperacillin/tazobactam was restored in the presence of piperacillin/BLI-489 combination.[65,66] BLI-489 at 4 mg/l in combinationwith piperacillin demonstrated a low probability of spontaneous resistance development in vitro for a panel of Gram-negativestrains encoding various types of β-lactamase enzymes, with the exception of P. aeruginosa.[67]

LK-157 is a tricyclic carbapenem inhibitor of class A and C β-lactamases. In combination with various antibiotics, it restored theactivity against ESBLs, except for CTX-M and KPC-producing strains.[68]

Oxapenems (AM-112–115) are new antimicrobials capable of β-lactamase inhibition with activity against class A, C and Denzymes, but results of studies on this compounds have not been published since 2004.[69,70] AM-114 and AM-115 displayedthe most potent activity against class A, comparable to that of clavulanic acid, while their activity against class C and D enzymeswas superior to that of clavulanic acid.[69] In another study, ceftazidime plus AM-112 was more effective than ceftazidime aloneagainst a strain of E. coli containing TEM-1 and CTX-M-1 β-lactamases.[70] An enhanced activity of oxapenems in combinationwith ceftazidime was also reported against P. aeruginosa strains and MRSA.[69]

NXL104 is a non-β-lactam compound that inhibits β-lactamases through the formation of a stable covalent carbamoyllinkage.[66] Recently, NXL104 has been tested in combination with cefepime, ceftazidime, ceftriaxone, mecillinam andmeropenem against 190 ESBL-producing E. coli and K. pneumoniae, 94 AmpC hyperproducing E. coli and eight AmpC/ESBLco-expressing E. coli. Remarkably, NXL104 restored susceptibility to the partner cephalosporins for all isolates tested.[71]

Table 5. Old and new β-lactamase inhibitors and specific activity against different classes of β-lactamases.

Inhibitor Class US FDA status

A B C D

Inhibitors with β-lactam structure

Clavulanic acid + - + + Approved

Tazobactam + - + + Approved

Sulbactam + - + + Approved

BLI-489 + ? + + Phase I†

BAL30376 ? + + ? Phase I†

LK-157 + ? + ? Preclinical

Oxapenems + ? + + Preclinical

Inhibitors without β-lactam structure

NXL104 + + + + Phase I and II†‡

ME1071 ? + ? ? Phase I (Japan)†

MK7655 + ? + ? Phase I‡

†Complete results not published.‡In combination with ceftaroline and ceftazidime, respectively.+: Active; -: Nonactive; ?: Unknown.Data from [101].

Although NXL104 does not inhibit MBLs, it can overcome the ceftazidime resistance engendered by AmpC enzymes in P.aeruginosa and other nonfermenting rods, even when these are completely derepressed.[72] Moreover, NXL104 atconcentrations of less than or equal to 4 mg/l protects ceftaroline from hydrolysis against 90 Enterobacteriaceae, 60 of whichwere ESBL-producers and 30 of which expressed high levels of AmpC-related β-lactamases.[73]

The NXL104/ceftazidime combination is currently undergoing clinical trials: NXL104/ceftazidime plus metronidazole versusmeropenem in complicated intra-abdominal infections and NXL104/ceftazidime versus imipenem/cilastatin in UTIs (trialsongoing).

Finally, MK-7655 is a novel compound active against class A and class C carbapenemases with a good in vitro and in vivoactivity in combination with imipenem.[74,75] In a Phase I randomized, double-blind, placebo-controlled study, MK-7655 wasshown to have a favorable pharmacokinetic profile when administered in combination with cilastatin and imipenem.[76]

Inhibitors Active against MBL

Reports on MBL production mostly involve nonfermenting rods such as P. aeruginosa and A. baumannii.[77] Nevertheless, afterthe spread of NDM-1 enzymes among E. coli and K. pneumoniae, MBL production among Enterobacteriaceae is also reaching acrisis level.[22] Two promising compounds, BAL30376 and ME1071, may restore activity of different β-lactams against MBLs-producers.

BAL30376 is a β-lactamase inhibitor composed of a siderophore monobactam (BAL19764), a bridged monobactam (BAL29880,which is a class C inhibitor) and clavulanic acid.[22] BAL30376 was active at 4 mg/l against MBL-producing strains and someisolates of Burkholderia cepacia and carbapenemase-producing A. baumannii. However, it was inactive against KPC-producingstrains.[78] In a recent study, BAL30376 has shown good in vitro activity against most species of Enterobacteriaceae, includingstrains that produced MBLs. The activity of BAL30376 against MBL-producers was largely attributable to the intrinsic stability ofthe monobactam BAL19764 towards these enzymes.[79]

ME107 is another new MBL-inhibitor that competitively inhibits both IMP-1 and VIM-2 enzymes. It significantly enhancedbiapenem activity in a concentration-dependent manner against MBL-producing P. aeruginosa.[80]

New Drugs in Old Classes Other than β-lactams

The development of novel compounds belonging to already known antimicrobial classes attempt to overcome resistance tocurrently used tetracyclines, aminoglycosides and polymyxins.

TP-434 is a fluorocycline with in vitro and in vivo activity against MDR Gram-negatives, except for P. aeruginosa.[81]

TP-434 is currently in Phase I development as an intravenous formulation and an oral formulation is in preclinical phasedevelopment.[82] ACHN-490 is a novel aminoglycoside currently undergoing a Phase II evaluation in complicated UTIs and isresistant to plasmid-mediated aminoglycoside-modifying enzymes.[30,83] ACHN-490 has shown better activity than currentlyused aminoglycosides against 82 carbapenem-resistant Enterobacteriaceae. However, it was not able to overcome NDM-1-mediated resistance and 16 of 17 NDM-1-producing isolates were resistant to ACHN-490 and currently usedaminoglycosides.[83] Antimicrobial activity of ACHN-490 has also been tested against clinical isolates of E. coli, K. pneumoniae,A. baumannii and P. aeruginosa.[84,85] ACHN-490 was active against most isolated strains of E. coli and K. pneumoniae,including MDR strains, and MICs for ACHN-490 were four-times lower than for amikacin.[85] The MICs of ACHN-490 against P.aeruginosa were similar to that of amikacin.[83] However, the reported MICs for A. baumannii show lower values than those ofcurrently used aminoglycosides.

CB-182,804 is a polymyxin B analog that has demonstrated high activity against colistin-susceptible and -resistant isolates.[86]

Colistin-susceptible strains that were resistant to all other available antimicrobials were found to be susceptible.[86] CB-182,804is currently in Phase I of clinical development.[86]

New Classes of Antimicrobials

New antimicrobials interacting with DNA as well as bacterial enzymes or specific antigens may represent the key to reachingwide and unexplored fields in the treatment of MDR bacteria. In addition, they may lead to a reduction in the administration ofcurrently used antimicrobials and decrease their selective pressure on resistance. FDA status and antimicrobial activity of novelantimicrobials against MDR Gram-negative strains is outlined in Table 6.

Table 6. US FDA status and antimicrobial activity of novel antimicrobials against multidrug-resistantGram-negative strains.

Drug US FDAstatus

Antimicrobial class In vitro activity against MDR Gram-negative bacteria

Escherichiacoli

Klebsiellapneumoniae

Pseudomonasaeruginosa

Acinetobacterbaumannii

Bis-indole antibiotics are compounds that enter bacterial cells and interact with DNA. The interaction resulted in the inhibition ofDNA and RNA synthesis and in the induction of the SOS response. Therefore, it is likely that the DNA-binding activity of thesecompounds is directly related to their mechanism of antibacterial action.[87] One of these compounds, MBX 1162, has shownremarkable in vitro activity against 13 and 12 isolates of MDR A. baumannii and ESBL-producing K. pneumoniae, respectively.Of note, it was also active against vancomycin-resistant enterococcus and MRSA.[88]

AN3365 is a novel boron-containing antibacterial, capable of inhibiting protein synthesis through the inhibition of tRNAsynthetase.[89] AN3365 demonstrated an excellent in vitro activity against various Gram-negative bacteria, includingciprofloxacin-resistant isolates, as well as a favorable pharmacokinetic profile in mice.[90–92]

LpxC is a deacetylase involved in the biosynthesis of lipopolysaccharide in the cell of Gram-negatives and it is a validated targetfor developing novel antimicrobial agents designed as potent LpxC inhibitors.[93] These agents have a novel mechanism ofaction and are active against MDR P. aeruginosa and MDR E. coli (MIC: <1 mg/l).[82] Moreover, they have the potential to beadministered orally as well as intravenously.[82]

The Rx-04 program represents a new approach to antibiotic research and development. It identified 1400 crystal structures ofknown and new antibiotics capable of binding the 50S ribosome, thereby defining an available antibiotic design space in a largeribosomal-binding site.[82] Among 100 new analogs, several showed activity against E. coli and P. aeruginosa, including MDRones.[94]

An interesting niche in antibiotic development is occupied by antimicrobial peptides. This group of molecules is produced by alltypes of living organisms and it is considered to be part of the host innate immunity. Their ability to kill MDR microorganisms hasgained them considerable attention and clinical interest.[95] One of these compounds, mastoparan, showed good activity againstboth colistin-susceptible and -resistant A. baumannii strains, suggesting that the mechanism of action of this antimicrobialpeptide may be different to that used by colistin.[96]

Finally, antimicrobial specificity may be selectively addressed by antibodies targeting unique bacterial antigens. KB001 is a high-affinity antibody fragment in development for the treatment of P. aeruginosa infections.[97] KB001 binds to the PcrV protein ofthe type III secretion system, which is responsible for delivery of exotoxins, and inhibits its activity, leading to a reduction in thepathogenicity of P. aeruginosa.[98]

Expert Commentary & Five-year View

The spread of MDR Gram negatives, such as ESBL or carbapenemase-producing Enterobacteriaceae and A. baumannii or P.aeruginosa is reaching a level of crisis due to the lack of available therapeutic options, with a relevant impact in terms ofmorbidity, mortality and healthcare-associated costs. The awareness of mechanisms and epidemiology of resistance is anecessary requirement to approach the development of new antimicrobials active against MDR strains. Bacteria continue todevelop resistance mechanisms turning back the clock to the preantibiotic era and the pace of antibiotic drug development hasslowed during the last decade. There is a critical need for new antimicrobials, with special emphasis on new anti-Gram-negativeagents. Among novel antimicrobials in development, compounds with anti-Gram-negative activity include β-lactamase inhibitors,cephalosporins, carbapenems and other compounds belonging to old and new classes of antimicrobials. Although fifth-generation cephalosporins acquired activity against MRSA, they offer no advantage against MDR Gram-negatives, with theexception of CXA-101. Some of the novel carbapenems are active against resistant Gram-positives, but when difficult Gram-negatives are involved, especially MBL-producers, they may lose their activity. Among novel compounds belonging to alreadyknown classes of antimicrobials, β-lactamase inhibitors are the most promising, as they might restore the activity of alreadyknown β-lactams against β-lactamase-producing strains, including class C, D and B β-lactamase-producing strains. However,

KB001 Phase II Antibody fragment - - + -

CB-182,804 Phase I Polymyxins + + + +

AN3665 Phase I Protein synthesisinhibitors + + + +

TP-434 Phase I Tetracyclines + + + +

MBXagents Preclinical Bis-indoles ? + ? ?

CHIR-090 Preclinical LpxC inhibitors + ? + ?

+: Active; -: Nonactive; ?: Unknown; MDR: Multidrug resistant.Data from [101].

their real clinical utility will be known only after results of clinical trials become available and, nevertheless, antibiotics of existingclasses are generally incremental improvements on existing agents and, as such, are subject to the same resistance. Therefore,novel classes of antibiotics with different mechanisms of action are urgently needed in the near future. Antibody fragments caninhibit virulence factors of P. aeruginosa reducing pathogenicity and are currently undergoing Phase II studies. Other novelcompounds with antimicrobial activity include bis-indoles, boron-containing antibacterial protein synthesis inhibitors, outermembrane synthesis inhibitors and antimicrobial peptides. However, they are still undergoing preclinical or Phase I studies andthey will not reach the market in the short term. Indeed, truly new therapeutic options against MDR strains are still far fromclinical practice and physicians are forced to rediscover old drug classes such as polymyxins and optimize the administration ofcurrently used antimicrobials. For example, a prolonged infusion of a high dose of cefepime, ceftazidime, doripenem andmeropenem seems to partially restore the efficacy of β-lactams against strains with decreased susceptibility. It is also crucialthat we improve our antibiotic resistance surveillance capacity to cope with the rapid, worldwide spread of emerging resistancemechanisms and clones. Appropriate control measures against the spread of MDR pathogens and antimicrobial stewardshipprograms in human medicine, agriculture and animal husbandry are also important principles to adopt not only until new drugsreach clinical practice, but also in the future, aiming to reduce the insurgence and spread of resistance against novelantimicrobials. The current Gram-negative resistance scenario may be soon defined in a similar manner to that of MRSA, withthe appearance of Enterobacteriaceae and nonfermenting rods resistant to all available β-lactams in clinical practice. Thedevelopment of new antimicrobials represents only a part of the adequate countermeasures to adopt, especially in the nearfuture. However, an appropriate and strategic resistance-based approach to the development of new compounds is mandatoryto reduce the spread of MDR and XDR pathogens.

Sidebar

Key Issues

The spread of resistance among Gram-positive and Gram-negative bacteria represents a growing challenge for thedevelopment of new antimicrobials.Especially for Gram-negatives, clinicians are facing a dramatic shortage in the availability of therapeutic options.Truly new therapeutic options against multidrug resistance (MDR) strains are still far from clinical practice and, until newdrugs reach clinical practice, one of the residual and effective strategies to treat infections caused by MDR Gram-negatives is the optimization in the use of already existing molecules and the rediscovery of old ones such as polymyxinsand fosfomycin.An appropriate and strategic resistance-based approach to the development of new compounds is mandatory to reducethe spread of MDR and extreme-drug resistance pathogens.Novel anti-Gram-negative agents include β-lactamase inhibitors, cephalosporins, carbapenems and other compoundsbelonging to old and new classes of antimicrobials.Although their real clinical utility will be known only after results of clinical trials become available, among novelcompounds belonging to already known classes of antimicrobials, β-lactamase inhibitors are the most promising, as theymight restore the activity of already known β-lactams against β-lactamase-producing strains, including class C, D and B β-lactamases-producing strains.Novel classes of compounds with antimicrobial activity include monoclonal antibodies, bis-indoles, boron-containingantibacterial protein synthesis inhibitors, outer membrane synthesis inhibitors and antimicrobial peptides, but thesecompounds are still undergoing preclinical or Phase I studies.Appropriate control measures against the spread of MDR pathogens and antimicrobial stewardship programs in humanmedicine, agriculture and animal husbandry are also important principles to adopt, aiming to reduce the insurgence andspread of resistance against novel antimicrobials.

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Financial & competing interests disclosure Matteo Bassetti serves on scientific advisory boards for Pfizer Inc., Merck Serono, Novartis, Shionogi & Co., Ltd. and Astellas Pharma Inc.; he hasreceived funding for travel or speaker honoraria from Pfizer Inc., Merck Serono, Novartis, GlaxoSmithKline, Gilead Sciences, Inc., Sanofi-Aventis,Cephalon, Inc., Bayer Schering Pharma, Janssen and Astellas Pharma Inc. The authors have no other relevant affiliations or financial involvementwith any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apartfrom those disclosed.No writing assistance was utilized in the production of this manuscript.

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